Method for differentiating mesenchymal stem cells into neural cells

ABSTRACT

A method for differentiating mesenchymal stem cells of bone marrow into neural cells comprises culturing the mesenchymal stem cells in a medium containing epidermal growth factor(EGF), basic fibroblast growth factor(bFGF) and hepatocyte growth factor(HGF), and the neural cells produced thereby can be employed for the treatment of a neural disease.

FIELD OF THE INVENTION

The present invention relates to a method for differentiatingmesenchymal stem cells in bone marrow into neural cells by culturingthem in a medium containing epidermal growth factor(EGF), basicfibroblast growth factor(bFGF) and hepatocyte growth factor(HGF), and acomposition for treating a neural disease comprising the neural cells asan active ingredient.

BACKGROUND OF THE INVENTION

Stem cells have the ability to divide indefinitely in culture and giverise to specialized cells constituting a tissue upon stimulation by aspecific differentiation stimulus.

Stem cells are divided into embryonic stem cells(ES cells) andtissue-specific stem cells depending on their differentiation potencies.ES cells are isolated from the inner cell mass(ICM) of embryos at theblastocyst stage and are pluripotent, i.e., they are capable ofdifferentiating into virtually every type of cells found in an organism.

In contrast, tissue-specific stem cells appear at a stage of organformation during the embryonic development and they are organ-specificand multipotent, i.e., they are generally committed to give rise tocells constituting a specific organ. These tissue-specific stem cellsremain in most of adult organs and perform the critical role ofcontinually replenishing the loss of cells occurring normally orpathologically. Representative tissue-specific stem cells includehematopoietic stem cells and mesenchymal stem cells present in bonemarrow. Hematopoietic stem cells give rise to various blood cells suchas erythrocytes and leukocytes; and mesenchymal stem cells, to the cellsof connective tissues, e.g., osteoblasts, chondroblasts, adipocytes andmyoblasts.

Recently, clinical applications of the stem cells have drawn anincreasing interest since the successful isolation of human embryonicstem cell. The most noticeable potential application of the stem cellsis their use as a perfect source of cell supply for a cell replacementtherapy. Hardly curable diseases, e.g., neurodegenerative disease suchas Parkinson's and Alzheimer's diseases, quadriplegia resulting fromspinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiacinfarction and liver cirrhosis, are caused by the disruption andpermanent functional disorder of the cells constituting an organ, andthe cell replacement therapy, wherein the loss of cells is replenishedfrom the outside, has been presented as an effective remedy.

However, notwithstanding the obvious benefit of the cell replacementtherapy, there exist many limitations in its clinical applications.Specifically, the conventional method, wherein fully differentiatedcells isolated from the tissues of a donor are transplanted into apatient, has the problem that it is difficult to obtain a sufficientamount of cells to be supplied to the patient. In order to solve thisproblem, cells of a specific tissue differentiated from an isolatedembryonic stem cell or differentiated cells from isolated andproliferated tissue-specific stem cells can be employed in a cellreplacement therapy.

Hitherto, it has been proven that mouse embryonic stem cells can bedifferentiated on a culture dish into various cells such ashematopoietic cells, myocardial cells, insulin-secreting pancreaticcells and neural cells. Further, several reports have demonstrated thattransplantation of the cells differentiated from stem cells is effectivein the treatment of a disease caused by the loss of cells. For instance,synthesis of myelin in a mouse increased when myelin-synthesizingoligodendrocytes differentiated from an embryonic stem cell weretransplanted into the mouse(Brustle et al., Science, 285: 754–756,1999). Blood sugar level was regulated by transplantinginsulin-secreting cells differentiated from an embryonic stem cell intoa diabetes mouse model (Soria et al., Diabetes, 49: 157–162, 2000).Further, dyscinesia caused by spinal cord injury was remediedsignificantly by transplanting neural cells differentiated from anembryonic stem cell into a mouse having spinal cord injury(McDonald etal., Nat. Med., 5(12): 1410–1412, 1999).

However, since the human embryonic stem cell has been successfullyisolated only recently and there is no report on the differentiation ofthe embryonic stem cell on a culture dish into other specific cells thanneural cells, clinical use of specific tissue cells differentiated froman embryonic stem cell in a cell replacement therapy still remains on alevel of possibility.

Further, since the efficiency of differentiation from an embryonic stemcell into target cells is low, there is a risk of an adverse side effectcaused by other cells mixed with the target cells duringtransplantation. Accordingly, there exists a need for the development ofa precise differentiation method for safer clinical application of thecells differentiated from embryonic stem cells.

On the other hand, in case when tissue-specific stem cells are employedin a cell replacement therapy, there is the problem that lowering of theproliferating ability of the cells or differentiation into unfavorablecells may occur during a long-term culture. Further, transplantation ofneural cells is required for the treatment of a neurodegenerativedisease such as Parkinson's disease. Since it is difficult to obtainneural stem cells directly from the patient, they are generally obtainedby culturing neural stem cells isolated from the brain tissue of deadfetus and differentiating them into neural cells. However, the use offetal brain invites the ethical problem as well as is limited byinsufficient supply, and may cause an immunological rejection. Further,most of neural stem cells are liable to differentiate into astrocytesrather than neurons.

Accordingly, if it is possible to differentiate mesenchymal stem cellsin patient's own bone marrow into neural cells to be used in a cellreplacement therapy, neural cells can be readily supplied and suchproblems as immunological rejection would not occur during treatment.

Hitherto, it has been considered that one kind of stem cellsdifferentiate only into the cells of a tissue belonging to a specificsystem. It was reported that mesenchymal stem cells formed in vitrocolonies in the presence of various growth factors such asplatelet-derived growth factor, basic fibroblast growth factor(bFGF),transforming growth factor-β (TGF-β) and epidermal growthfactor(EGF)(Kuznetsov et al., Br. J. Haematol., 97: 561, 1997; and vanden Bos C. et al., Human Cell, 10:45, 1997), and about one-third ofinitially attached cells had a multipotency, thereby differentiatinginto connective tissue cells such as osteoblasts, chondroblasts andadipocytes(Pittenger M F et al., Science, 284: 143, 1999). Further,Ferrari G. et al. reported that bone marrow is a source of myogenicprecursor cells that form new muscles(Science, 279: 1528, 1998).

Recent studies reported that mesenchymal stem cells can alsodifferentiate into the cells of the neural system. For instance,Sanchez-Ramos et al. reported that mesenchymal stem cells differentiatedinto neurons and astrocytes upon culture in the presence of retinoicacid and brain-derived neurotrophic factor(BDNF)(Exp. Neurology, 164:247–256, 2000). Dale Woodbury et al. reported that mesenchymal stemcells in bone marrow differentiated into neural cells in the presence ofantioxidants such as β-mercaptoethanol and dimethyl sulfoxide(DMSO)(J.Neuro. Res., 61: 364–370, 2000). However, the use of strongdifferentiation-inducing agents such as DMSO may cause a problem in aclinical application.

The present inventors have endeavored to discover materials that arehighly safe and capable of differentiating stem cells in bone marrowinto neural cells, and have discovered that HGF promotes thedifferentiation of mesenchymal stem cells of bone marrow into neuralcells, and also that the addition of EGF and bFGF to a culture medium,together with HGF, significantly enhances both the differentiation ofstem cells into neural cells and the amplification of the resultingneural cells.

It has been reported that EGF and bFGF stimulate the differentiation ofneural stem cells into neurons or astrocytes when they are added to aserum-free medium for culturing neural stem cells separated from thebrain tissue (Melissa et al., Exp. Neurology, 158: 265–278, 1999).

HGF has been reported to enhance the viability of neurons in hippocampusand mesencephalon, and induce the growth of neurite in neocorticalexplant(Hamanoue M et al., J. Neurosci. Res., 43: 554–564, 1996).Further, in the peripheral nervous system, it functions as an existencefactor for motoneurons(Ebens A et al., Neuron, 17: 1157–1172, 1996), andinvolves in the growth and existence of sensory neurons andparasympathetic neurons (Fleur Davey et al., Mol. Cell Neurosci., 15:79–87, 2000).

However, it has never been reported that mesenchymal stem cells can bedifferentiated into neural cells by culturing them in a mediumcontaining EGF, bFGF and HGF.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod for differentiating mesenchymal stem cells or mononuclear cellsin bone marrow into neural cells.

It is another object of the present invention to provide neural cellsdifferentiated by said method and a pharmaceutical composition fortreating a neural disease comprising the neural cells as an activeingredient.

It is a further object of the present invention to provide a method fortreating a neural disease in a mammal, which comprises administering theneural cells produced by the above method to a subject in need thereof.

In accordance with one aspect of the present invention, therefore, thereis provided a method for differentiating mesenchymal stem cells in bonemarrow into neural cells, which comprises culturing the mesenchymal stemcells in a medium containing epidermal growth factor(EGF), basicfibroblast growth factor(bFGF) and hepatocyte growth factor(HGF).

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, which respectivelyshow:

FIG. 1: a photomicrograph(×100; hereinafter, same magnification isapplied) of the attached cells derived from mononuclear cells of bonemarrow cultured for 4 weeks on a medium containing 10 ng/ml EGF, 20ng/ml bFGF and 20 ng/ml HGF;

FIG. 2: a photomicrograph of the neural cells differentiated frommononuclear cells of bone marrow cultured for 8 weeks on a mediumcontaining 10 ng/ml EGF, 20 ng/ml bFGF and 20 ng/ml HGF;

FIGS. 3A and 3B: photomicrographs of a neuron and an astrocyte,respectively, which are isolated from the neural cells of FIG. 2;

FIGS. 4A to 4C: the results of immunocytochemical staining ofdifferentiated neural cells of FIG. 2, wherein FIG. 4A is NSE-positivecells; FIG. 4B, NeuN-positive cells and FIG. 4C, GFAP-positive cells;

FIGS. 5A to 5C: photomicrographs of osteoblasts, chondroblasts andadipocytes, respectively, differentiated from mesenchymal stem cells;

FIG. 6: a photomicrograph of mesenchymal stem cells taken immediatelyafter its inoculation on a medium containing 10 ng/ml EGF, 20 ng/ml bFGFand 20 ng/ml HGF;

FIG. 7: a photomicrograph of neural cells differentiated from themesenchymal stem cells cultured for 8 weeks on a medium containing 10ng/ml EGF, 20 ng/ml bFGF and 20 ng/ml HGF; and

FIGS. 8A to 8C: the results of immunocytochemical staining of thedifferentiated neural cells of FIG. 7, wherein FIG. 8A is NSE-positivecells; FIG. 8B, NeuN-positive cells and FIG. 8C, GFAP-positive cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for differentiating mesenchymalstem cells isolated from bone marrow into neural cells, which comprisesculturing the mesenchymal stem cells in a medium containing epidermalgrowth factor (EGF), basic fibroblast growth factor (bFGF) andhepatocyte growth factor (HGF).

Further, neural cells differentiated by said method and a pharmaceuticalcomposition for treating a neural disease comprising the neural cells asan active ingredient are also provided.

As used herein, the term “neural cells” refers to the nerve-like cellsincluding neurons, astrocytes and microglias.

In order to differentiate mesenchymal stem cells into neural cells, itis preferred to culture the mesenchymal stem cells in a cell culturemedium containing 1 to 1,000 ng/ml, preferably, 5 to 10 ng/ml of EGF, 1to 1000 ng/ml, preferably, 10 to 20 ng/ml of bFGF and 1 to 1000 ng/ml,preferably, 5 to 20 ng/ml of HGF for more than one week. It is morepreferable to culture the stem cells for more than 4 weeks. About 4weeks after the initiation of culture, neural cell colonies consistingof several cells are formed and, after about 8 weeks, a massive amountof neural cells are produced by the continuous growth and proliferationof the neural cell colonies.

In contrast, if the mesenchymal stem cells are cultured in the samemedium but lacking HGF, the cells can not differentiate into neuralcells. Further, if they are treated with HGF only, early differentiationoccurs, thereby preventing proliferation. This result suggests that, inthe inventive process of culturing mesenchymal stem cells to obtainneural cells, EGF and bFGF stimulate the cells to proliferate and HGFstimulates the stem cells to differentiate into neural cells. Further, asufficient amount of neural cells cannot be obtained when only one ofthe factors is employed.

Once the neural cells are completely differentiated and proliferatedafter 8-week culture, they can further proliferate without losing theirunique characteristics as neural cells even when treated only with EGFand bFGF. On the other hand, if the fully differentiated andproliferated neural cells are subsequently treated with HGF only, thecells differentiate continuously without proliferation and,consequently, the number of cells decreases. Accordingly, afterculturing for 8 weeks, it is preferred to allow the neural cells toproliferate by further culturing them in a medium containing EGF andbFGF.

According to the present method using EGF, bFGF and HGF, more than 80%of the total differentiated cells are neural cells, which comprises 60to 80%, preferably 65 to 75%, of neurons and 20 to 40%, preferably 25 to35%, of astrocytes, based on the number of cells, and contains nomicroglia.

In the present invention, mesenchymal stem cells are preferably obtainedfrom human bone marrow. Mononuclear cells derived from bone marrowcontains hematopoietic and mesenchymal stem cells, and when they arecultured for 1 to 2 weeks, hematopoietic stem cells easily differentiateinto mature blood cells. Accordingly, the stem cells proliferatingthereafter are mesenchymal stem cells, which continue to prolifereateafter 20 passages of subculture. The mesenchymal stem cellsdifferentiate into various connective tissue cells includingosteoblasts, chondroblasts and adipocytes.

In addition, massive production of neural cells can also be achievedwhen the mononuclear cells of bone marrow including the mesenchymal stemcells are used in the method of the present invention, in place of theisolated mesenchymal stem cells.

The present method is advantageous especially in terms of safety, sincethe neural cells are differentiated from the mesenchymal stem cells byemploying as inducers the specific proteins present in a human body,i.e., EGF, bFGF and HGF, without using a harmful differentiation inducersuch as DMSO. Further, since a sufficient amount of neural cellsnecessary for the treatment of a disease can be produced from patient'sown bone marrow, a clinical application thereof would be viable owing toready availability of neural cells and reduced risk of immunologicalrejections.

The neural cells differentiated from mesenchymal stem cells inaccordance with the present invention can be used as an activeingredient of a cell composition for a cell replacement therapy for aneural disease. Non-limiting examples of neural diseases, which can betreated by using the neural cells of the present invention, includeneurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease, Pick's disease, Huntington's disease, amyotrophic lateralsclerosis and ischemic brain disease. The inventive neural cells canalso be used in the treatment of various diseases caused by unusual lossof neural cells as well as dyscinesia caused by a spinal cord injury.

A cell composition for preventing or treating neural diseases can beprepared by mixing the neural cells differentiated by the inventivemethod with a pharmaceutically acceptable excipient or carrier, or bydiluting it with a pharmaceutically acceptable diluent in accordancewith any of the conventional procedures. Examples of suitable carriers,excipients, and diluents are lactose, dextrose, sucrose, sorbitol,mannitol, xylitol, erythritol, maltitol, starches, gum acacia,alginates, gelatin, calcium phosphate, calcium silicate, cellulose,methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone,water, methylhydroxybenzoates, propylhydroxy-benzoates, talc, magnesiumstearate and mineral oil. The formulations may additionally includefillers, anti-agglutinating agents, lubricating agents, wetting agents,flavors, emulsifiers, preservatives and the like. The cell compositionof the present invention may be formulated so as to provide quick,sustained or delayed release of the active ingredient after theiradministration to a mammal by employing any of the procedures well knownin the art. Thus, the formulations may be in the form of a sterileinjectable solution, suspension, emulsion, solution and the like,wherein a sterile injectable solution is preferred.

Accordingly, the present invention also provides a method for treating aneural disease in a mammal, which comprises administering the neuralcells produced by the inventive method to a subject in need thereof inan amount effective for treating the disease.

The neural cells produced by the inventive method may be injected intothe body of a patient by any of the conventional methods. For instance,the method of Douglas Kondziolka(D. Kondziolka et al., Neurology 55:556–569, 2000) may be used. Specifically, the cranium of the patient isexcised to create an opening having a diameter of about 1 cm, and a HBSS(Hank's balanced salt solution) containing neural cells is injected inabout three spots. The injection is carried out by a syringe having along needle and a stereotactic frame for injecting the desired cellsolution into a deep part of the brain at a correct position. The cellcomposition of the present invention can be administered via variousroutes including transdermal, subcutaneous, intravenous andintramuscular introduction, surgical stereotactic introduction,intralesional introduction by vascular catheterization.

Typical unit dose of the neural cells may range from 1×10⁶ to 1×10⁹cells and they can be administered every week or every month. However,it should be understood that the amount of the active ingredientactually administered ought to be determined in light of variousrelevant factors including the disease to be treated, the severity ofthe patient's symptom, the chosen route of administration, and the age,sex and body weight of the individual patient; and, therefore, the abovedose should not be intended to limit the scope of the invention in anyway.

The following Examples are intended to further illustrate the presentinvention without limiting its scope.

Further, percentages given below for solid in solid mixture, liquid inliquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis,respectively, and all the reactions were carried out at roomtemperature, unless specifically indicated otherwise.

EXAMPLE 1 Isolation of Mononuclear Cells in Bone Marrow

About 10 ml of bone marrow was taken from the pelvis of each of healthyvolunteers and stored in glass tubes containing heparin. 30 ml ofphosphate buffered saline(PBS) was added to 10 ml of bone marrow, and 20ml of the resulting mixture was slowly transferred onto 10 ml ofFicoll-Paque™ plus solution(1.077 g/ml, Amersham Pharmacia Biotech), andsubjected to density gradient centrifugation at 2000 rpm for 20 minutes.The mononuclear cell layer at the interface between the top layer andFicoll-Paque™ plus layer was recovered and subject to centrifugation at1800 rpm for 5 minutes to obtain mononuclear cells.

EXAMPLE 2 Culture of Mononuclear Cells

The mononuclear cells obtained in Example 1 were inoculated at a densityof 1×10⁶ cells/cm² to a culture flask containing a basal medium. Theflask was incubated at 37° C., 5% CO₂. After 4 hours, the flask waswashed with fresh basal medium to remove non-attached cells. The basalmedium was Williams' E medium(Gibco BRL) containing 3.5 μM ofhydrocortisone(Sigma), 50 ng/ml of linoleic acid(Sigma Co.) mixed withfatty acid-free bovine serum albumin(Gibco BRL) at an equal molar ratio,0.1 μM CuSO₄.5H₂O (Sigma), 50 pM ZnSO₄.7H₂O (Sigma), 3 ng/ml H₂SeO₃(Sigma), 1.05 mg/ml NaHCO₃(Sigma Co.), 1.19 mg/ml HEPES(Sigma), 100 U/mlpenicillin(Gibco BRL), 10 mg/ml streptomycin(Gibco BRL) and 25 μg/mlamphotericin(Gibco BRL).

EXAMPLE 3 Differentiation of Mononuclear Cells into Neural Cells

In order to confirm whether the mononuclear cells obtained in Example 2differentiate into neural cells, the mononuclear cells were cultured at37° C., 5% CO₂ on a basal medium containing 10 ng/ml of epidermal growthfactor(Gibco BRL), 20 ng/ml of basic fibroblast growth factor(R&DSystems) and 20 ng/ml of hepatocyte growth factor(R&D Systems)(“differentiating medium”). The differentiating medium was replenishedtwo times a week.

About 4 weeks after, neural cell colonies appeared and continuouslyproliferated(see Table 1 and FIG. 1).

About 8 weeks after, neuron-form cells consisting of long projectionssuch as axons and short projections such as dendrites, andastrocyte-form cells consisting only with short dendrites were observed(see FIGS. 2 and 3). Further, after 8 weeks, the cells proliferated withtheir shapes unaltered, even if cultured on a basal medium containingonly EGF and bFGF(see Table 2).

However, when the mesenchymal stem cells were cultured in a mediumcontaining only EGF and bFGF, the cells did not differentiate intoneural cells. Further, when the mesenchymal stem cells were cultured ina medium containing HGF only, the cells differentiated early and,accordingly, they neither grew nor proliferated(see Table 1).

TABLE 1 Number of cells/ml of culture after 8-week culture ofmesenchymal stem cells No. of Treatment Treatment with inoculated Nogrowth with Treatment with EGF, bFGF cells factor HGF EGF and bFGF andHGF 7.5 × 10⁷ Not Not 1 × 10⁵ 2 × 10⁵ proliferated proliferated

TABLE 2 Proliferation of cells after 4- or 8-week culture of neuralcells differentiated from mesenchymal stem cells by 8-week culture on amedium containing EGF, bFGF and HGF (No. of inoculated cells: 1 × 10⁵)Treatment Treatment with Treatment with with EGF EGF, bFGF No growthfactor HGF and bFGF and HGF After 4 Not proliferated Not proliferated 2× 10⁵ 2 × 10⁵ weeks After 8 Not proliferated Not proliferated 5 × 10⁵ 1× 10⁵ weeks

EXAMPLE 4 Immunocytochemistry

The neural cells obtained in Example 3, which were differentiated fromthe mononuclear cells of bone marrow by culturing on a medium containingEGF, bFGF and HGF for 8 weeks, were attached on a 1 cm² cover glass at adensity of 1×10⁴ cells/cm². The cells were washed with 0.1 M phosphatebuffer for 5 minutes, fixed with 0.1 M phosphate buffer containing 4%paraformaldehyde for 15 minutes, and washed twice with 0.1 M phosphatebuffered saline(PBS). The cells were treated with 0.1 M PBS containing1% BSA and 0.2% Triton X-100 for 5 minutes, and then, reacted for 16hours with first antibodies; mouse anti-human neuron-specificenolase(NSE)(Chemicon Inc.), mouse anti-human neuron-specific nuclearprotein (NeuN)(Chemicon Inc.), mouse anti-human β-tubulin III (SigmaCo.) and mouse anti-human glial fibrillary acidic protein(GFAP)(SigmaCo.).

Upon completion of the reaction with the first antibodies, the remainingantibodies were removed and the cells were washed twice with 0.1 M PBScontaining 0.5% BSA each for 15 minutes. A secondary antibody, rabbitanti-mouse IgG (Sigma Co.) was added thereto and incubated for 30minutes. The cells were washed with 0.1 M PBS containing 0.5% BSA eachfor 5 minutes. The reaction was carried out for 30 minutes by employingVectastain Elite ABC kit(Vector Laboratory Inc.) containingavidin-biotin. The cells were washed twice with 0.1 M phosphate buffereach for 5 minutes, DAB (3,3′-diaminobenzidine tetrahydrochloridedehydrate, Sigma Co.) was added thereto as a color developing substrate,and the mixture was allowed to react for 5 minutes. The reaction wasstopped by treating the reactants with 0.1 M phosphate buffer for 5minutes and washing them twice with the buffer each for 5 minutes. Theresulting reactants were dried and washed with distilled water for 5minutes. The cells were dehydrated and fixed by treating sequentiallywith distilled water, and 70%, 80%, 95% and 100% ethanol.

The results of the above immunocytochemical staining are shown in FIGS.4A to 4C, wherein the differentiated cells exhibit positive results forneuronal markers NeuN, NSE and β-tubulin III, and astroglial markerGFAP. These results show that the cells were differentiated into neuronsand astrocytes, as judged by their biochemical as well as morphologicalcharacteristics. However, the cells were negative for microglial markerOX-42, demonstrating that the mononuclear cells did not differentiateinto microglia.

The proportions of neurons(which is positive for NeuN and NSE) andastrocytes(which is positive for GFAP) in the cells differentiated fromthe mononuclear cells of bone marrow by culturing on a medium containingEGF+bFGF+HGF or EGF+bFGF for 8 weeks, were examined and shown in Table3.

TABLE 3 Negative NSE NeuN GFAP cells EGF + bFGF ab. 0.9% Ab. 0.8% ab.1.2% ab. 89% EGF + bFGF + HGF ab. 56% Ab. 75% ab. 24% ab. 20%

As can be seen from Table 3, about 80% of the total differentiated cellsare neural cells when cultured on a medium containing EGF+bFGF+HGF for 8weeks. The neural cells consisted of about 70% neurons and about 30%astrocytes.

EXAMPLE 5 Isolation and Culture of Mesenchymal Stem Cell

In order to see whether the mesenchymal stem cells in the mononuclearcells of bone marrow would differentiate into neural cells, themononuclear cells were cultured and mesenchymal stem cells were isolatedtherefrom. The mesenchymal stem cells were examined for their capabilityto differentiate into various cells, as follows.

The mononuclear cells cultured as in Example 2 were inoculated in aculture flask containing DMEM (Gibco BRL) supplemented with 10% FBS(fetal bovine serum), at a density of 1×10³ cells/cm². The cells werecultured at 37° C. under an atmosphere of 5% CO₂. After 1˜2 weeks, theproliferated cells were subjected to a subculture, and the proliferationcontinued after 20 passages of subculture.

Mononuclear cells obtained from bone marrow include mature leukocytes,lymphocytes, scleroblasts, chondrocytes, muscle cells, fibroblasts,adipocytes as well as stem cells to be differentiated into these cells,said stem cells being divided into hematopoietic and mesenchymal stemcells. The hematopoietic stem cells, which give rise to blood cells suchas erythrocytes, leukocytes and lymphocytes, can not proliferate buteasily differentiate into mature blood cells in a general culturemedium. Accordingly, it can be seen that the cells proliferatingcontinuously as above are mesenchymal stem cells.

In order to confirm whether the proliferating cells are indeedmesenchymal stem cells, the cells were treated with various cytokinesand chemical agents and their differentiation into various connectivetissue cells, including osteoblasts, chondroblasts and adipocytes, wasexamined in accordance with the method of Pittenger et al., Science,284: 143–147, 1999.

In order to differentiate the stem cells into osteoblasts, the cellswere treated with 100 mM dexamethasone, 10 mM β-glycerol phosphate and50 nM ascorbate-2-phosphate and 10% FBS.

Further, in order to confirm the capability of the stem cells todifferentiate into chondroblasts, cultured stem cells were centrifugedto obtain cell pellets, which were treated with 100 nM dexamethasone and10 ng/ml TGF-β 3 in the absence of serum.

The differentiation of the stem cells into adipocytes was induced bytreating the stem cells with 0.5 mM 1-methyl-3-isobutylxanthine, 1 mMDexamethasone, 10 g/ml insulin, 100 nM indomethacine and 10% FBS.

Osteoblasts were examined by alkaline phosphatase staining(Jaiswal etal., J. Cell Biochem., 64(2): 295–312, 1997); chondroblasts, by type IIcollagen RT-PCR(Mackay et al., Tissue Eng., 4(4): 415–428, 1998) andstaining with toluidine blue; adipocytes, by staining with oil red O.

Consequently, as can be seen from FIGS. 5A, 5B and 5C, positive resultswere observed under a light microscope in all of the samples. Theseresults demonstrate that the mesenchymal stem cells cultured andproliferated in vitro still maintain the properties of stem cellscapable of differentiating into various connective tissue cells such asosteoblasts, chondroblasts and adipocytes.

EXAMPLE 6 Differentiation of Mesenchymal Stem Cells into Neural Cells

To examine whether the mesenchymal stem cells isolated in Example 5 candifferentiate into neural cells, the mesenchymal stem cells werecultured for 8 weeks in a medium supplemented with EGF, bFGF and HGF,according to the procedure of Example 3.

Consequently, like the differentiation of neural cells from mononuclearcells in bone marrow, neural cell colonies were formed after 4 weeks andthe neural cells grew and proliferated continuously until the 8th week.FIGS. 6 and 7 illustrate photomicrographs of the cells taken immediatelyafter inoculation and after 8-week culture, respectively.

After 8th week, the cells continuously proliferated while maintainingthe morphological characteristics of neural cells even when they weretreated only with EGF and bFGF.

Also, immunocytochemical staining was carried out with thedifferentiated cells, according the procedure of Example 4.

Consequently, like the results obtained for the neural cells derivedfrom mononuclear cells of bone marrow, the differentiated cells weretested positive for neural markers NeuN, NSE and β-tubulin III andastroglial marker GFAP. Accordingly, it was confirmed that mesenchymalstem cells differentiate into both neurons and astrocytes(see FIGS. 8Ato 8C). FIGS. 8A to 8C show the results of immunocytochemical staining,FIG. 8A representing NSE-positive cells; FIG. 8B, NeuN-positive cells;and FIG. 8C, GFAP-positive cells.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

1. A method for differentiating mesenchymal stem cells into neuralcells, which comprises culturing the mesenchymal stem cells in a mediumcontaining epidermal growth factor (EGF), basic fibroblast growth factor(bFGF) and hepatocyte growth factor (HGF).
 2. The method of claim 1,wherein the mesenchymal stem cells are cultured in a medium containing 1to 1000 ng/ml of EGF, 1 to 1000 ng/ml of bFGF and 1 to 1000 ng/ml of HGFfor more than one week.
 3. The method of claim 2, wherein the culturingis carried out for more than 4 weeks.
 4. The method of claim 1, whereinthe mesenchymal stem cells are cultured in a medium containing EGF, bFGFand HGF for more than one week and the resulting differentiated neuralcells are allowed to proliferate in a medium containing EGF and bFGF. 5.The method of claim 1, wherein the mesenchymal stem cells are isolatedfrom human bone marrow.
 6. The method of claim 1, wherein mononuclearcells isolated from bone marrow and containing mesenchymal stem cellsare used as a source of mesenchymal stem cells.
 7. The method of claim1, wherein the neural cells contain neurons and astrocytes.